Enzymatic Assay of β-Phosphoglucomutase
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of β-Phosphoglucomutase.
2. SCOPE
This procedure applies to all products that have specification for the enzymatic activity of β -Phosphoglucomutase at Sigma-Aldrich Saint Louis.
3.1.NADP+ - β -Nicotinamide Adenine Dinucleotide Phosphate, Oxidized
3.2.NADPH- β -Nicotinamide Adenine Dinucleotide Phosphate, Reduced
3.3.Purified Water - water from a deionizing system, resistivity ~ 18mΩ•cm at 25ºC
3.4.Unit Definition – One unit is defined as the amount of enzyme, which converts 1μmol of β-D-glucose-1-phosphate to β-D-glucose-6-phosphate per minute at 37°C and pH 7.0.
4. DISCUSSION
β-D-Glucose-1-Phosphate Phosphoglucomutase >β-D-glucose-6-Phosphate
β-D-Glucose-6-Phosphate+NADP+ Glucose-6-Phosphate Dehydrogenase >6-Phosphogluconate+NADPH+H+
5. RESPONSIBILITIES
It is the responsibility of all trained Analytical Services personnel to follow this protocol as written.
6. SAFETY
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:
T = 37ºC, Abs340nm, pH 7.0, Light Path= 1cm
7.2 METHOD
Spectrophotometric Rate Reaction
7.3 REAGENTS
7.3.1. 300 mM HEPES/40 mM Potassium Chloride/4 mM Magnesium Chloride, pH 7.0 at 37ºC (BUFFER)
Prepare a solution containing 72 mg/ml of HEPES, Sigma-Aldrich Product Number such as H3375, with 3 mg/ml of Potassium Chloride, Sigma-Aldrich Product Number such as P4504, and 0.4% v/v of 1M Magnesium Chloride, Sigma-Aldrich Product Number such as M1028, in purified water. Adjust pH to 7.0 at 37ºC with 1N NaOH.
7.3.2. 10.0 mM α-D-Glucose-1, 6-Diphosphate, Potassium Salt (G-1,6-DiPO4)
Prepare a solution containing 5 mg/ml of α-D-Glucose-1, 6-Diphosphate, Potassium Salt, Sigma-Aldrich Product Number such as G6893, in purified water. Glucose 1,6-Diphosphate is required in order to obtain maximum activity.
7.3.3. 4.8 mM β-D-Glucose-1-Phosphate (G-1-PO4)
Weigh 80 mg – 85 mg of β -D-Glucose-1-Phosphate, Disodium, Dicyclhexylammonium Sigma-Aldrich Product Number such as G7920. Weigh-up is corrected for water, solvent and dicyclhexylammonium.
7.3.4. 60 mM β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized (NADP+)
Prepare a solution of 60 mM β-Nicotinamide Adenine Dinucleotide Phosphate, Oxidized, Disodium Salt, Sigma-Aldrich Product Number such as N0505, in purified water. Weigh-up is corrected for water, solvent and sodium. Adjust pH to 7.0 with Sodium Bicarbonate, ACS reagent, Sigma-Aldrich Product Number such as S8875.
7.3.5. 10 mM Potassium Phosphate (Enzyme Diluent)
Prepare a solution containing 0.87 mg/ml of Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number such as P5379, with 0.67 mg/ml of Potassium Phosphate, Diabasic, Anhydrous Sigma-Aldrich Product Number such as P8281, in purified water. Adjust pH to 7.0 with KOH at 37ºC.
7.3.6. Glucose-6-phosphate Dehydrogenase (G6PDH)
Immediately before use, prepare a 1000 un/ml solution of Glucose-6-Phosphate Dehydrogenase in cold Reagent 7.3.5 (Enzyme Diluent) using Glucose 6-phosphate Dehydrogenase, Type XV, from Baker’s Yeast, Sigma-Aldrich Product Number such as G6378.
7.3.7 β-Phosphoglucomutase (Enzyme)
Immediately prior to use, prepare a stock solution of 5mg solid/ml in cold reagent 7.3.5. Then immediately dilute 0.10 ml to 15.1 ml in cold Reagent 7.3.5.
7.3.8. Cocktail
Mix the following reagents in a 50 ml beaker:
| Reagent 7.3.3 (G-1-PO4) | 90.0 mg to 100 mg |
| Reagent 7.3.1 (BUFFER) | 20 ml |
| Reagent 7.3.2 (G-1,6-DiPO4) | 4 ml |
| Purified Water | 12 ml |
Adjust pH to 7.0 at 37ºC with 0.1N KOH or 0.1N HCl
7.3.9. ENZYMATIC
Pipette (in milliliters) the following reagents into suitable cuvettes:
| Test1 | Test2 | Test3 | Blank | |
| Cocktail | 3.00 | 3.00 | 3.00 | 3.00 |
| Reagent 7.3.5 (Enzyme Diluent) | 0.010 | 0.005 | ------- | 0.050 |
| Reagent 7.3.4 (NADP+) | 0.100 | 0.100 | 0.100 | 0.100 |
| Reagent 7.3.6 (G6PDH) | 0.100 | 0.100 | 0.100 | 0.100 |
Mix by inversion and equilibrate to 37ºC with a thermostatted spectrophotometer. Then add:
| Reagent 7.3.7 (Enzyme) | 0.040 | 0.045 | 0.050 | ------- |
Immediately mix by inversion and record the increase in Abs340nm for 100 minutes. Record the maximum linear rate for a 5-minute interval (Δ Abs340nm/ minute) for all test and blank reactions.
7.4 CALCULATIONS
7.4.1. ΔAbs340nm/minute = ΔAbs340nm/minute (Test) - ΔAbs340nm/minute (Blank)
| 7.4.2. | Units/ml= | (ΔAbs340nm/minute )x(3.25)x df |
| (6.22)(0.05) |
6.22 = Extinction coefficient of NADPH
3.25= Total volume (in milliliters) of Enzymatic Reaction Mixture
0.05= Volume (in milliliters) of enzyme solution (Reagent 7.3.7) used in enzymatic reaction mixture
df= dilution factor of enzyme solution
| 7.4.3. | Units/mg solid = | Units/ml |
| mg solid/ ml |
| 7.4.4. | Units/mg protein = | Units/ml |
| mg protein/ ml |
7.5 FINAL ASSAY CONTENTRATION
In a 3.25ml reaction mix the final concentrations are 154 mM HEPES, 20.5 mM Potassium Chloride, 2.1 mM Magnesium Chloride, 2.3 mg – 2.6 mg of β-D-Glucose-1-Phosphate, 1.02mM α-D-Glucose-1, 6-Diphosphate, Potassium Salt, 0.15mM Potassium Phosphate, 1.8mM β-NADP, 30.8 units of Glucose-6-phosphate Dehydrogenase and 0.41-0.51 μg of β-Phosphoglucomutase.
8.1. Procedure replaces SPGLUC29.
8.2. Nakamura, K. et al. J. Ferment. Bioeng., (1998) 85, 350-353.
8.3. Biozyme Laboratories International, Ltd. AP213
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